ez007012 at chip.ucdavis.edu (Taru Kosonen) wrote:
>> I'm trying to determine the halflife of some relatively low abundant
> transcrpits in human skin fibroblasts using the pulse-chase method with
> 3-H -uridine. Anybody familiar with this method? My specific concern is
> about how to take the reincorporation/recycling of the label into account?
>> Any help/hints are greatly appreciated. You can also answer directly by
> email: takosonen at ucdavis.edu>> Thanks. Taru Kosonen
> Dept. of Nutrition
> UC DAVIS
Getting a sharp end to the pulse is the big problem, which is why many
people use actinomycin D to stop all new mRNA synthesis at the end of
the labelling. In the absence of inhibitors, I've used a wash with PBS
containing 100 ug/ml uridine and 35mM glucosamine (as a sink for UTP)
and then further chase incubation in growth medium with these
supplements.
Keith Leppard